U.S. patent application number 11/883661 was filed with the patent office on 2008-12-25 for uses of dinucleotide polyphosphate derivatives.
Invention is credited to Natalya Lozovaya, Andrew David Miller, Julian Alexander Tanner, Michael Wright.
Application Number | 20080319184 11/883661 |
Document ID | / |
Family ID | 34307930 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080319184 |
Kind Code |
A1 |
Miller; Andrew David ; et
al. |
December 25, 2008 |
Uses of Dinucleotide Polyphosphate Derivatives
Abstract
The present invention provides the use of analogues and
derivatives of dinucleoside polyphosphates with formula (I) or a
pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for use in one or more of: the treatment of ischemia,
inducing ischemic tolerance, modulating cerebral ischemia, to delay
the onset of a hypoxic depolarisation stage when ischemic events
are initiated; as a neurological protection agent; as a tissue
protection agent; the treatment of pain; and the treatment of
inflammation, wherein X, is selected from wherein X.sup.1 and
X.sup.2 are independently selected from H, Cl, Br and F; each Y is
independently selected from S and O; each Z is independently
selected from --CX.sup.3X.sup.4--,--NH--,--O--; wherein X.sup.3 and
X.sup.4 are selected from H, Cl, Br and F; B1 and B2 are
independently selected from adenine, guanine, xanthine, thymine,
uracil, cytosine and inosine; S.sub.1 and S.sub.2 are independently
selected from ribose, open chain ribose, 2'-deoxyribose,
3'deoxyribose and arabinofuranoside. V is selected from 0, 1, 2, 3,
4 and 5; W is selected from 0, 1, 2, 3, 4 and 5; and V plus W is an
integer from 2 to 6.
Inventors: |
Miller; Andrew David;
(London, GB) ; Wright; Michael; (London, GB)
; Tanner; Julian Alexander; (Hong Kong, HK) ;
Lozovaya; Natalya; (London, GB) |
Correspondence
Address: |
SUZANNAH K. SUNDBY;SMITH, GAMBRELL & RUSSEL, LLP
1130 Connecticut Avenue, NW, Suite 1130
WASHINGTON
DC
20036
US
|
Family ID: |
34307930 |
Appl. No.: |
11/883661 |
Filed: |
February 1, 2006 |
PCT Filed: |
February 1, 2006 |
PCT NO: |
PCT/GB06/00343 |
371 Date: |
August 27, 2008 |
Current U.S.
Class: |
536/26.26 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 25/28 20180101; A61P 9/10 20180101; C07H 21/00 20130101; A61P
25/14 20180101; A61P 21/00 20180101; A61P 25/00 20180101; C07H
21/04 20130101; C07H 21/02 20130101; A61P 1/04 20180101; A61P 25/04
20180101; A61P 19/06 20180101; A61P 1/16 20180101; A61P 17/00
20180101; A61P 19/02 20180101; A61K 31/7084 20130101; A61P 29/00
20180101 |
Class at
Publication: |
536/26.26 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 3, 2005 |
GB |
0502250.4 |
Claims
1. Use of a compound of formula (1): ##STR00012## or a
pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for use in one or more of: treatment of ischemia, as a
neurological protection agent; as a tissue protection agent;
treatment of pain; and treatment of inflammation; wherein X, is
selected from ##STR00013## wherein X.sup.1 and X.sup.2 are
independently selected from H, Cl, Br and F; each Y is
independently selected from S and O; each Z is independently
selected from --CX.sup.3X.sup.4--, --NH--, --O--; wherein X3 and X4
are selected from H, Cl, Br and F; B.sub.1 and B.sub.2 are
independently selected from adenine, guanine, xanthine, thymine,
uracil, cytosine and inosine; S.sub.1 and S.sub.2 are independently
selected from ribose, 2'-deoxyribose, 3'deoxyribose,
arabinofuranoside and ring opened forms thereof. V is selected from
0, 1, 2, 3, 4 and 5; W is selected from 0, 1, 2, 3, 4 and 5; and V
plus W is an integer from 2 to 6.
2. Use of a compound of formula (1) according to claim 1 wherein at
least one of B.sub.1 and B.sub.2 is adenine.
3. Use of a compound of formula (1) according to claim 1 wherein
B.sub.1 and B.sub.2 are both adenine.
4. Use of a compound of formula (1) according to claim 1 wherein
S.sub.1 and S.sub.2 are the same.
5. Use of a compound of formula (1) according to claim 4 wherein
S.sub.1 and S.sub.2 are ribose.
6. Use of a compound of formula (1) according to claim 1 wherein
each Z is 0.
7. Use of a compound of formula (1) according to claim 1 wherein V
is 2.
8. Use of a compound of formula (1) according to claim 1 wherein W
is 2.
9. Use of a compound of formula (1) according to claim 1 wherein X
is --CX.sup.1X.sup.2--.
10. Use of a compound of formula (1) according to claim 1 wherein
and X.sup.1 and X.sup.2 are both H.
11. Use of compound of formula (1) according to claim 1, or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for use in one or more of: (a) treatment of diseases and
medical conditions associated with P2-receptors; (b) treatment of
diseases and medical conditions associated with Al adenosine
receptors; (c) moderating the activity of P2-receptors; (d)
moderating the activity of Al adenosine receptors; and (e) for
modulating K+ influx via G protein-gated inwardly rectifying
K.sup.+ (GIRK) channels in mammalian cells.
12. A compound selected from: (a) App.sub.spA ##STR00014## (b)
A.sub.diolppCH2ppA.sub.diol ##STR00015## (c) AppNHpppU
##STR00016##
13. (canceled)
Description
[0001] The present invention relates to the use of analogues and
derivatives of dinucleoside polyphosphates.
[0002] Dinucleoside polyphosphates are a group of compounds
comprising two nucleoside moieties linked by a polyphosphate
bridge. Dinucleoside polyphosphates form an important family of
compounds and are thought to have both intracellular and
extracellular biological roles..sup.1,2
[0003] One dinucleoside polypeptide of particular interest is
diadenosine 5',5'''-P.sup.1,P.sup.4-tetraphosphate (Ap.sub.4A).
Ap.sub.4A is thought to function in cellular responses to cell
proliferation and environmental stresses in prokaryotes and lower
eukaryotes, as well as to play a role in extracelluar signalling in
higher eurkaryotes..sup.3,4 It has also been reported that
Ap.sub.4A may have protective effects in the cortex and midbrain in
defined rat models of stroke and Parkinson's disease..sup.5
[0004] A number of synthetic methods have been reported for the
preparation of dinucleoside polyphosphates and attempts have been
made to study their biological roles in more detail..sup.6,7
Despite this, and the fact that many of these compounds are widely
found in nature and have been known for a number of years,.sup.3 it
has proved difficult to define the biological functions of such
compounds. Indeed, the confusion over the role of such compounds
has led to an ambiguous suggestion that they could act as either
"friend or foe"..sup.8 In general there have been considerable
difficulties in obtaining results that are sufficiently
reproducible to correlate biological functions in vivo or ex vivo
with the presence of dinucleoside polyphosphates, reasons for this
are not clear but could be related to hydrolytic
lability..sup.3
[0005] Attempts to study and use dinucledside polypeptides have
also been hampered by the frequent difficulties encountered in
isolating and purifying such compounds from natural sources. For
example, diadenosine polyphosphates (Ap.sub.nA; n=2-6) appear to be
highly unstable to specific enzymatic and non-specific hydrolysis
in biological fluids and tissue samples..sup.3,10
[0006] The present invention alleviates the problems of the prior
art.
[0007] In one aspect the present invention provides the use of a
compound of formula (1):
##STR00001##
[0008] or a pharmaceutically acceptable salt thereof,
[0009] in the manufacture of a medicament for use in one or more
of:
[0010] treatment of ischemia,
[0011] as a neurological protection agent;
[0012] as a tissue protection agent;
[0013] treatment of pain; and
[0014] treatment of inflammation;
[0015] wherein X, is selected from
##STR00002##
[0016] wherein X.sup.1 and X.sup.2 are independently selected from
H, Cl, Br and F;
[0017] each Y is independently selected from S and O;
[0018] each Z is independently selected from
--CX.sup.3X.sup.4--, --NH--, --O--;
[0019] wherein X.sup.3 and X.sup.4 are selected from H, Cl, Br and
F;
[0020] B.sub.1 and B.sub.2 are independently selected from adenine,
guanine, xanthine, thymine, uracil, cytosine and inosine;
[0021] S.sub.1 and S.sub.2 are independently selected from ribose,
2'-deoxyribose, 3'deoxyribose, arabinofuranoside and ring opened
forms thereof.
[0022] V is selected from 0, 1, 2, 3, 4 and 5;
[0023] W is selected from 0, 1, 2, 3, 4 and 5; and
[0024] V plus W is an integer from 2 to 6.
[0025] We have found that by our choice of X and Z groups we
provide use of compounds (and novel compounds) which give
persistent and reproducible biological outcomes in the presence of
biological fluids and tissue samples. Thus a use and novel
compounds are provided which allow for reproducible effects in
vivo.
[0026] For ease of reference, these and further aspects of the
present invention are now discussed under appropriate section
headings. However, the teachings under each section are not
necessarily limited to each particular section.
PREFERRED ASPECTS
[0027] X
[0028] X is selected from
##STR00003##
wherein X.sup.1 and X.sup.2 are independently selected from H, Cl,
Br and F.
[0029] In one aspect X is --NH--.
[0030] In one aspect X is
##STR00004##
[0031] In one aspect X is --CX.sup.1X.sup.2--.
[0032] In one aspect at least one of X.sup.1 and X.sup.2 is H.
[0033] In one aspect at least one of X.sup.1 and X.sup.2 is Cl.
[0034] In one aspect at least one of X.sup.1 and X.sup.2 is Br.
[0035] In one aspect at least one of X.sup.1 and X.sup.2 is F.
[0036] Preferably both X.sup.1 and X.sup.2 are H.
[0037] Preferably X is --CX.sup.1X.sup.2-- and X.sup.1 and X.sup.2
are both H.
[0038] Y
[0039] Each Y is independently selected from S and O;
[0040] In one aspect at least one Y is S.
[0041] In one aspect each Y group is S.
[0042] In one aspect at least one Y is O.
[0043] Preferably each Y group is O.
[0044] Z
[0045] Each Z is independently selected from
--CX.sup.3X.sup.4--, --NH--, --O--
wherein X.sup.3 and X.sup.4 are selected from H, Cl, Br and F;
[0046] In one aspect at least one Z is --CX.sup.3X.sup.4--
[0047] In one aspect each Z is --CX.sup.3X.sup.4--.
[0048] In one aspect at least one of X.sup.3 and X.sup.4 is H.
[0049] In one aspect at least one of X.sup.3 and X.sup.4 is Cl.
[0050] In one aspect at least one of X.sup.3 and X.sup.4 is Br.
[0051] In one aspect at least one of X.sup.3 and X.sup.4 is F.
[0052] Preferably both X.sup.3 and X.sup.4 are H.
[0053] Preferably Z is --CX.sup.3X.sup.4-- and X.sup.3 and X.sup.4
are both H.
[0054] In one aspect at least one Z is --NH--.
[0055] In one aspect each Z is --NH--.
[0056] In one aspect at least one Z is --O--.
[0057] Preferably each Z is --O--.
[0058] B.sub.1 and B.sub.2
[0059] B.sub.1 and B.sub.2 are independently selected from adenine,
guanine, xanthine, thymine, uracil, cytosine and inosine;
[0060] In one aspect at least one of B.sub.1 and B.sub.2 is
uracil.
[0061] In one aspect at least one of B.sub.1 and B.sub.2 is
guanine.
[0062] Preferably at least one of B.sub.1 and B.sub.2 is
adenine.
[0063] Preferably at least one of B.sub.1 and B.sub.2 is adenine
and the other of B.sub.1 and B.sub.2 is guanine.
[0064] Preferably at least one of B.sub.1 and B.sub.2 is adenine
and the other of B.sub.1 and B.sub.2 is uracil.
[0065] Preferably B.sub.1 and B.sub.2 are both adenine.
[0066] S.sub.1 and S.sub.2
[0067] S.sub.1 and S.sub.2 are independently selected from ribose,
2'-deoxyribose, 3'deoxyribose, arabinofuranoside and ring opened
forms thereof.
[0068] Preferably at least one of S.sub.1 and S.sub.2 is
ribose.
[0069] Preferably at least one of S.sub.1 and S.sub.2 is a ring
opened form of ribose.
[0070] Preferably at least one of S.sub.1 and S.sub.2 is ribose and
the other of S.sub.1 and S.sub.2 is a ring opened form of
ribose.
[0071] Preferably S.sub.1 and S.sub.2 are the same.
[0072] Preferably S.sub.1 and S.sub.2 are ribose.
[0073] V and W
[0074] V is selected from 0, 1, 2, 3, 4 and 5.
[0075] W is selected from 0, 1, 2, 3, 4 and 5.
[0076] V plus W is an integer from 2 to 6, that is the sum of V and
W may be 2, 3, 4, 5 or 6.
[0077] Preferably V is 2.
[0078] Preferably W is 2.
[0079] Preferably V plus W is 4.
Other Aspects and Features
[0080] Preferably, the compound of formula (1) is:
##STR00005##
[0081] Preferably the compound of formula (1) is:
##STR00006##
[0082] Preferably the compound of formula (1) is:
##STR00007##
[0083] Preferably the compound of formula (1) is:
##STR00008##
[0084] In a further aspect the present invention provides the use
of compound of formula (1) as described herein, or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for use in one or more of:
[0085] (a) treatment of diseases and medical conditions associated
with P2-receptors;
[0086] (b) treatment of diseases and medical conditions associated
with A1 adenosine receptors;
[0087] (c) moderating the activity of P2-receptors;
[0088] (d) moderating the activity of A1 adenosine receptors;
and
[0089] (e) for modulating K+ influx via G protein-gated inwardly
rectifying K.sup.+ (GIRK) channels in mammalian cells.
[0090] In a further aspect, the present invention provides a
compound selected from:
[0091] (a) App.sub.spA
##STR00009##
[0092] (b) A.sub.diolppCH2ppA.sub.diol
##STR00010##
[0093] (c) AppNHpppU
##STR00011##
[0094] Preferably the compound is App.sub.spA.
[0095] Preferably the compound is A.sub.diolppCH2ppA.sub.diol.
[0096] Preferably the compound is AppNHpppU.
[0097] Ischemia
[0098] In one aspect, the present invention relates to the
treatment of ischemia and ischemic related diseases and disorders.
These treatments may include inducing ischemic tolerance,
modulating cerebral ischemia and delaying the onset of a hypoxic
depolarisation stage when ischemic events are initiated. Ischemic
conditions occur when there is an inadequate supply of blood to an
organ or a part of a human or animal body. As a consequence of this
inadequate supply of blood, the organ or part of the body is
deprived of oxygen and nutrients, such as glucose. This can result
in the organ or part of the body being damaged. For example, if the
blood supply to any portion of the central nervous system (CNS) is
interrupted, the nerve cells (or neurons) of that portion of the
CNS will rapidly degenerate.
[0099] In particular, the present invention may relate to the use
of compounds in the manufacture of a medicament for the treatment
of the following disorders: focal ischemia; global ischemia;
cerebral ischemia; neuronal cell ischemia, such as the neuronal
cell ischemia associated with spinal injuries and head trauma;
myocardial ischemia; cardiovascular diseases, selected from the
group: hypertension, angina, stable and unstable angina, Prinzmetal
angina, arrhythmia, thrombosis, embolism, and congestive heart
failure including chronic or acute congestive heart failure; or a
disease characterised by ischemia of lower legs due to peripheral
vascular disease, including intermittent claudication; a disease
characterised by spasms of smooth muscle, selected from the group:
spasms of the ureter, spasms of the bladder, uterine cramps, and
irritable bowel syndrome; or in the prevention of vasoconstriction
and/or ischemic tissue damage during a surgical procedure, selected
from the group: bypass grafts, angiography, angioplasty, organ
preservation during transplant, hypertensive crisis or post
operative hypertension.
[0100] Neurological Diseases and Disorders
[0101] The present invention may be useful in the treatment of
neurological diseases and disorders, in particular, in the
treatment of neuronal cells. Such treatments include the treatment
of brain trauma, brain or cerebrovascular ischemia,
neurodegenerative diseases, poisoning of neuronal cells, and the
preservation of neuronal grafts.
[0102] Neurodegenerative diseases are a group of disorders
characterised by changes in the normal neuronal function, which may
lead to neuronal death (most of these diseases are associated,
especially in the later stages, with severe neuronal loss). These
neurodegenerative diseases may include amyotrophic lateral
sclerosis, Alzheimer's disease, Parkinson's disease and
Huntington's disease.
[0103] Pain
[0104] In another aspect, the present invention may be useful in
the treatment of pain. Such treatments may include the treatment of
the pain associated with joint conditions (such as rheumatoid
arthritis and osteoarthritis), pain associated with cancer,
post-operative pain, postpartum pain, the pain associated with
dental conditions (such as dental caries and gingivitis), the pain
associated with bums (including sunburn), the treatment of bone
disorders (such as osteoporosis, hypercalcaemia of malignancy and
Paget's disease), the pain associated with sports injuries and
sprains.
[0105] Inflammation
[0106] In another aspect, the present invention may relate to the
treatment of inflammation. Inflammation may be caused by a variety
of conditions, so for example, the present invention may relate to
the treatment of arthritis, myocarditis, encephalitis, transplant
rejection, systemic lupus erythematosis, gout, dermatitis,
inflammatory bowel disease, hepatitis, or thyroiditis.
[0107] Stress
[0108] In another aspect, the present invention may relate to the
treatment of chemical and/or environmental stress. In particular,
the present invention may relate to the use of compounds to induce
neurological preconditioning. Following administration of suitable
compounds, such neurological preconditioning enables the
neurological tissue to tolerate and/or survive levels of chemical
and/or environmental stress which would normally prove lethal. This
use of compounds described in the present invention may relate to
of these compounds to elicit nitric oxide (NO), which can act as a
mediator in the preconditioning of tissues to chemical and/or
environmental stress.
[0109] The present invention will now be described in further
detail by way of example only with reference to the accompanying
figures in which:
[0110] FIG. 1 shows the synthesis of AppCH.sub.2ppG;
[0111] FIG. 2 shows the synthesis of AppNHpppU;
[0112] FIG. 3 shows the synthesis of
A.sub.diolppCH.sub.2ppA.sub.diol
[0113] FIG. 4 shows a summary diagram of orthodromically (top 2)
and antidromically (bottom 2) induced population spikes,
illustrating electrode positions;
[0114] FIG. 5 shows the effect of increasing amounts of
AppCH.sub.2ppA on orthodromically induced population spikes (FIG.
5A), antidromically induced population spikes (FIG. 5B) and
excitatory postsynaptic currents, EPSCs, (FIG. 5C);
[0115] FIG. 6 shows the influence of
pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and
AppCH.sub.2ppA on orthodromic spikes;
[0116] FIG. 7 shows the influence of cyclopentyl teophylline (CPT)
and AppCH.sub.2ppA on orthodromic spikes;
[0117] FIG. 8 shows the effect of .alpha.,.beta.-methylene-ATP on
orthodromic spikes;
[0118] FIG. 9 shows the effect of increasing amounts of ATP.gamma.S
on orthodromic spikes;
[0119] FIG. 10 shows the influence of diinosine tetrahydrophosphate
(Ip.sub.4I) and AppCH.sub.2PpA on orthodromic spikes;
[0120] FIG. 11 shows the influence of diinosine tetrahydrophosphate
(Ip.sub.4I) and AppCH.sub.2ppA on antidromic spikes;
[0121] FIG. 12 shows the influence of
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, (PTIO) and
AppCH.sub.2ppA on orthodromic spikes;
[0122] FIG. 13 shows the shows the influence of AppCH.sub.2ppA on
orthodromic spikes at 22.degree. C.;
[0123] FIG. 14 shows the shows the influence of AppCH.sub.2ppA on
orthodromic spikes at 36.degree. C.
[0124] The present invention will now be described in further
detail in the following examples.
EXAMPLES
[0125] Electrospray mass spectroscopy (ES-MS) carried on a Bruker
Esquire 3000 machine set to 100% fragment strength. Samples were
applied in 1:1 acetonitrile:water containing 0.1% acetic acid.
Proton and phosphorous NMR spectra were recorded on a 400 MHz
Bruker Ultrashield, with samples in D.sub.2O at 300K. 64 scans were
used for proton spectra, 1024 scans for phosphorous. For
simplicity, only those .sup.1H NMR signals particularly useful for
compound identification have been described.
Preparation of Compounds
[0126] AppCH.sub.2ppG
[0127] 10.times.1 ml portions of LysU reaction mixture was made up
in aliquots. This mixture contained 2 mM L-lysine, 10 mM
MgCl.sub.2, 160 .mu.M ZnCl.sub.2, and 6U of pyrophosphatase in 50
mM Tris-HCl buffer, pH 8.0..sup.7,11 Nucleotides were added to 8 mM
ATP and 4 mM GMPPCP (.alpha.,.beta.-methylene-guanosine
5'-triphosphate), the mixtures were vortexed and LysU added to 9
.mu.M concentration (dimer). The mixes were then incubated at
38.degree. C. and the reaction monitored by HPLC using a 2 ml
SOURCE 15Q (Amersham Biosciences) ion exchange column, packed in 50
mM Tris-HCl buffer (pH 8.0) and eluted with a 0-0.5 M gradient of
salt over 5 mins at 2 ml/min..sup.9 After 25 minutes the ATP and
GMPPCP peaks (previously seen at 5.5 min and 5.2 min) were lost and
had been replaced with an Ap.sub.4A peak at 7.2 min and the target
at 6.8 min. Continued incubation converted the Ap.sub.4A to
Ap.sub.3A (5.1 min) after 1 hour, without degradation of the
target. AppCH.sub.2ppG was purified using a 60 ml SOURCE Q column
packed in water, eluted with a 0-2M gradient of TEAB
(triethylammonium hydrogencarbonate buffer) over 30 mins at 8
ml/min, and lyophilised for storage at -20.degree. C. .sup.9,12
SoQ/NaCl HPLC showed a single peak and the product has an ES-MS
(M-H) of 848.9 m/z. .sup.1H NMR: 8.39 (1H, s, 8-H-AD), 8.15 (1H, s,
2-H-Ad), 8.03 (1H, s, 8-H-Gu), 6.04 (1H, s, 1'-H-rib[Ad]), 5.82
(1H, s, 1'-H-rib[Gu]), 2.46 (2H, t, O--CH.sub.2--O), 2-H-Gu not
seen as presumably labile, .sup.31P NMR: 8.74 (2P, m, .beta.-P),
-10.63 (2P, m, .alpha.-P). Yield was 90%+
[0128] AppNHpppU
[0129] LysU synthesis of AppNHpppU would require NH substituted
adenosine tetraphosphate, which is not available. Therefore, we
used a chemical coupling based on the dehydration agent EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide)..sup.13 AMPPNHP
(adenosine 5'-(.beta.,.gamma.-imido) triphosphate, 50 mg) and UDP
(uridine diphosphate, 150 mg) were dissolved 2 M HEPES pH 6.5 with
75 mM MgCl.sub.2 in 10.times.1 ml aliquots. 400 mg of EDC was added
to each aliquot, and the mixtures incubated at 37.degree. C., again
monitoring by SoQ/NaCl HPLC (UDP 3.8 min, AMPPNHP 4.7 min). After
12 h, two product peaks were seen at 6.1 min and 7.0 min. They were
extracted with the SoQ/TEAB column and lyophilised. .sup.9,12 Both
products showed a single band by SoQ/NaCl HPLC, the 6.1 min had an
ES-MS (M-H) of 788.6 m/z and was identified as Up.sub.4U, whilst
the 7.0 min had 890.7 m/z which matches ApppNHppU. ApppNHppU
.sup.1H NMR: 8.52 (1H, s, 8-H-Ad), 8.21 (1H, s, 2-H-Ad), 7.87 (1H,
s, 6-H-Ur), 6.08 (1H, s, 1'-H-rib[Ad]), 5.90 (2H, d 1'-H-rib[Ur]
and 5-H-Ur), 3-H-Ur not seen (presumably labile), O--NH--O
obscured, .sup.31p NMR: three close bands seen of complex
multiplicity, -10.89, -10.97 and -11.25. The latter is tentatively
identified as an overlay of .alpha.-P, .beta.-P and .epsilon.-P.
Up.sub.4U .sup.1H NMR: 7.91 (2H, s, 6-H-Ur), 5.93 (4H, s, 1'-H-rib
and 5-H-Ur) .sup.31P NMR: a single band (d m) is seen at -11.46
ppm. Yield was 45% with respect to initial AMPPNHP (though some
starting material was recovered).
[0130] A.sub.diolppCH.sub.2ppA.sub.diol
[0131] AppCH.sub.2ppA (100 mg, previously made by LysU coupling
from ATP and AMPPCP [.alpha.,.beta.-methylene-adenosine
5'-triphosphate]).sup.9 was dissolved in 2 ml distilled water. 150
.mu.l of 0.3 M aqueous sodium periodate was added, followed after
10 mins, with 50 .mu.l of 0.5 M aqueous sodium borohydride
(warning: H.sub.2 evolved). The reactions were monitored by
SoQ/NaCl HPLC as normal, with the 5.8 min peak of AppCH.sub.2ppA
up-shifting to 6.2 min on oxidation to the dialdehyde and falling
to 4.4 min on reduction to the diol. Separation by SoQ/TEAB
.sup.9,12 gave two main bands, which were extracted and
lyophilised. Both showed single peaks by SoQ/NaCl HPLC with the
greater product having a ES-MS (M-H) of 836.9 m/z and the lesser
585.7 m/z. These match A.sub.diolppCH.sub.2ppA.sub.diol and
A.sub.diolppCH.sub.2pp respectively.
A.sub.diolppCH.sub.2ppA.sub.diol .sup.1H NMR: 8.34 (2H, s, 8-H-Ad),
8.13 (2H, s, 2-H-Ad), 5.96 (2H, s, 1'-H-rib), 3.97 (4H, s,
2'-H-rib), 3.80 (4H, s, 3'-H-rib), 3.70 (4H, s, rib-CH.sub.2--O),
2.31 (2H, t, O--CH.sub.2--O) .sup.31P NMR: 7.17 (2P, q, .beta.-P),
-11.16 (2P, d, .alpha.-P). A.sub.diolppCH.sub.2pp .sup.1H NMR: 8.41
(1H, s, 8-H-Ad), 8.22 (1H, s, 2-H-Ad), 6.01 (1H, s, 1'-H-rib), 4.01
(2H, s, 2'-H-rib), 3.74 (4H, m, 3'-H-rib and rib-CH.sub.2--O), 2.36
(2H, t, O--CH.sub.2--O) .sup.31P NMR: 7.50 (1P, m, .beta.-P), 6.45
(1P, q d, .gamma.-P), -10.56 (1P, d, .delta.-P), -11.21 (1P, d m,
.alpha.-P). Yield was 80%.
[0132] Biological Tests
[0133] The following biological data was acquired in order to
determine the effects and mechanism of dinucleoside polyphosphate
analogues. Critically, where compounds are shown to be P2 and/or A1
receptor agonists, then they would be expected to possess an
impressive range of potential therapeutic properties. In the past
25 years there have been many studies suggesting that P2 and/or A1
receptor agonists used in both the central nervous system (CNS) and
peripheral nervous system (PNS) will facilitate or synergise with
the actions of a wide variety of CNS active drugs that include
analgesics, antipsychotics, antidepressants, anxiolytics,
nootropics/cognition enhancers and the various agents effective in
stroke related CNS damage. Furthermore, such receptor agonists also
appear to be potent neurological compounds in their own right.
[0134] Against Stroke and Ischaemia
[0135] Chemical agents acting as A1 receptor agonists appear to
promote stable neuronal membrane potentials that result in the
inhibition of neuronal excitability and excitatory amino acid (EAA)
release..sup.22 Blockade of EAA release thus prevents the
neurotoxic sequelae associated with activation of
N-methyl-D-aspartate (NMDA) receptor. A1 receptor agonists can also
reduce stroke related cell death and hippocampal
neurodegeneration..sup.22
[0136] Against Epilepsy
[0137] Chemical agents acting as A1 receptor agonists reduce
epileptic seizure activity induced by a variety of chemical and
electrical stimuli in animal models..sup.23,24 In electrically
kindled seizure models, A1 receptor agonists are anticonvulsants
that reduce seizure severity and duration without significantly
altering seizure threshold..sup.23
[0138] Against Neurodegeneration
[0139] There is wide acceptance that neuronal hyper-excitability
associated with ischaemia, hypoxia and epilepsy also underlies the
neurodegenerative processes associated with aging. Chemical agents
acting as P2 and/or A1 receptor agonists reduce EAA neurotoxicity
and the resultant changes in calcium homeostasis that lead to nerve
cell death may reflect an acute manifestation of more subtle, long
term changes that are associated with Alzheimer's Disease (AD) and
Parkinson's Disease (PD)..sup.25 Agonists are potent
anti-inflammatory agents.sup.26 acting to inhibit free radical
production and may thus provide additional benefit in providing
potential AD treatments over and above direct effects on
neurotransmitter-mediated neuronal events. A1 receptor agonists can
reduce the high affinity state of striatal Dopamine (DA) D1
receptors..sup.27 Functionally, the A1 agonist blocks DA D1
receptor-mediated locomotor activation in reserpinized mice.
Alternatively, agonists can attenuate peri-oral dyskinesias induced
by selective DA D1 activation in rabbits. This dynamic
inter-relationship between dopaminergic and purinergic systems in
the neurochemistry of psychomotor function offers new possibilities
for the amelioration of dopaminergic dysfunction via A1 receptor
modulation.
[0140] Against Wakefulness
[0141] Direct administration of chemical agents acting as A1
agonists into the brain elicits an EEG profile similar to that seen
in deep sleep, manifested as an increase in REM sleep with a
reduction in REM sleep latency that results in an increase in total
sleep..sup.28 A1 selective agonists may suppress slow wave sleep
(SWS) and paradoxical sleep (PS) prior to eliciting an increase in
SWS.
[0142] Against Pain
[0143] The application of ATP to sensory afferents results in
hyper-excitability and the perception of intense pain. The
nucleotide can also induce nociceptive responses at local sites of
administration and can facilitate nociceptive responses to other
noxious stimuli..sup.29 The pronociceptive actions of ATP are
mediated via P2X receptors present on sensory afferents and in the
spinal cord. Homomeric P2X3 and heteromeric P2X2/3 receptors are
highly localized on the sensory nerves that specifically transmit
nociceptive signals..sup.30 ATP is released from a number of cell
types (e.g. sympathetic nerves, endothelial cells, visceral smooth
muscle) in response to trauma.sup.31 and there is a substantive
body of evidence that activation of P2X3 receptors may initiate and
contribute to the peripheral and central sensitization associated
with visceral nociception..sup.31 P2X3 receptor expression is
up-regulated in sensory afferents and spinal cord following damage
to peripheral sensory fibers..sup.32 Thus the development of
selective, bio-available P2X3 receptor antagonists may be
anticipated to provide novel compounds for the treatment of
pain.
[0144] In contrast, the administration of chemical agents acting as
A1 receptor agonists provides pain relief in a broad spectrum of
animal models (e.g., mouse hot plate test, mouse-tail flick assay,
rat formalin test, mouse abdominal constriction
assay..sup.29,33,34,35 A1 agonists are also effective in relieving
neuropathic pain in rat models,.sup.36 and inhibit pain-associated
behaviour elicited by spinal injection of substance P and the
glutamate agonist, NMDA. In mechanistic terms, A1 receptor agonists
are known to inhibit the release of glutamate into the spinal fluid
and also reduce cerebrospinal fluid levels of substance P in
rat..sup.29,37,38 Glutamate is a key mediator of the abnormal
hyper-excitability of spinal cord dorsal horn neurons (central
sensitization) that is associated with states of clinical
pain..sup.39 Substance P is another key mediator of nociceptive
responses..sup.29,37,38 A1 agonists have also shown utility in
relieving human pain..sup.38 Spinal administration of A1 agonist
relieves allodynia in a neuropathic pain patients without affecting
normal sensory perception. Infusion improves pain symptoms in
clinical pain models reducing spontaneous pain, ongoing
hyperalgesia and allodynia in patients with neuropathic pain. In
addition, low dose infusions of agonists during surgery may reduce
the requirement for volatile anesthetic and for post-operative
opioid analgesia..sup.37,40
General Methods
[0145] Hippocampal Slice Preparation
[0146] This study was carried out on 21-day old Wistar rats
(WAG/GSto, Moscow, Russia). After rapid decapitation, rat brains
were immediately transferred to a Petri dish with chilled
(4.degree. C.) solution of the composition 120 mM NaCl, 5 mM KCl,
26 mM NaHCO.sub.3, 2 mM MgCl.sub.2 and 20 mM glucose. Calcium salts
were omitted to reduce possible neuronal damage. The solution was
constantly bubbled with 95%O.sub.2/5%CO.sub.2 gas mixture to
maintain pH7.4. Hippocampal slices (300-400 .mu.M thick) were cut
manually with a razor blade along the alveolar fibres to preserve
the lamellar structure of excitatory connections. During
preincubation and experiments, the slices were kept fully submerged
in the extracellular solution, pH7.4, comprised of 135 mM NaCl, 5
mM KCl, 26 mM NaHCO.sub.3, 1.5 mM CaCl.sub.2, 1.5 mM MgCl.sub.2,
and 20 mM glucose, subjected to continuous bubbling with 95%
O.sub.2/5% CO) at 30-31.degree. C. 25-50 mM picrotoxin (RBI,
Natick, Mass., USA) was also included into the extracellular
solution during experiments to suppress the inhibitory activity of
interneurons. Electrophysiological measurements were recording
after at least 2 h of preincubation.
[0147] Electrophysiological Measurements
[0148] Excitatory postsynaptic currents (EPSCs) were recorded by a
standard whole-cell patch clamp technique in the CA1 subfield of
the hippocampus in response to stimulation of the Schaffer
collateral/commissural pathway. To prevent the spread of electrical
activity from area CA3, mini-slices were prepared by making a cut
orthogonal to the stratum pyramidale extending to the mossy fibre
layer. The intracellular solution, pH 7.2, for patch pipettes was
comprised of 100 mM CsF (Merck, Darmstadt, FRG), 40 mM
NaH.sub.2PO.sub.4, 10 mM HEPES-CsOH, 10 mM Tris-HCl. 2-3 mM
N-(2,6-dimethyl-phenylcarbamoylmethyl)-triethylammonium bromide
(QX-314; Tocris Cookson, Bristol, UK) was routinely added to the
intracellular solution to block voltage-gated sodium conductances.
Patch pipettes were pulled from soft borosilicate glass on a
two-stage horizontal puller. When fire-polished and filled with the
intracellular solution, they had a resistance of 2-3M.OMEGA.. To
visualise cell bodies of CA1 pyramidal neurones, the stratum oriens
and alveus were removed with a saline jet from a micropipette.
Currents were digitally sampled at 400 ms intervals by a 12-digit
ADC board, filtered at 3 kHz, and data stored on a hard disk for
further analysis. Access resistance was monitored throughout the
experiments and ranged typically from 6 to 9M.OMEGA.. Data from
cells where access resistance changed by more than 25% during the
experiment were discarded. Extracellular field potentials were
recorded using Ni/Cr electrodes. The population spikes were
digitised and stored on computer disk. The effects of receptor
agonists of antagonists were measured as the mean ratio I/I.sub.o
where I was the current under the substances action and I.sub.o was
the current in control saline. To stimulate the Schaffer
collateral/commissural pathway input, a bipolar Ni/Cr electrode was
positioned on the surface of the slice. Current pulses (10-100 pA)
of 0.1-1 ms duration were delivered through the isolated stimulator
HG 203 (Hi-Med, London, UK) at 0.066-0.2 Hz.
Experiment 1
[0149] The time course of the changes of amplitude in
orthodromically induced population spikes (FIG. 5A), antidromically
induced population spikes (FIG. 5B) and excitatory postsynaptic
currents, EPSCs, (FIG. 5C) in a CA1 zone of rat hippocampal slices
prepared in accordance with the general procedure was measured.
Over time, increasing amounts of Ap.sub.2CH.sub.2p.sub.2A was
applied to the rat hippocampal slice. Thus, 1.9 .mu.m of
Ap.sub.2CH.sub.2p.sub.2A was applied after 10 mins, this was
increased to 3.7 .mu.m after 14 mins, and to 7.4 .mu.m after 18
mins.
[0150] The electrode positions used to induce the orthodromically
(top 2) and antidromically (bottom 2) induced population spikes in
this experiment are illustrated in FIG. 4. AppCH.sub.2ppA was found
to produce reproducibly fast and reversible inhibition of
orthodromically evoked field potentials in all synaptic pathways
(FIG. 4) in hippocampus including CA3-CA1 synapses (FIG. 5A). To
the right of the time course in FIG. 5A is shown the original
traces of population spikes (five-fold averaged) corresponding with
points 1 (control) and 2 (Ap.sub.2CH.sub.2p.sub.2A effect) in the
time course.
[0151] In contrast to the orthodromically evoked field potentials,
the amplitudes of antidromic spikes (here and below CA3-CA1
synapses) (FIG. 5B) as well as EPSCs, recorded in CA1 pyramidal
neuron, (FIG. 5C) remained unchanged. EPSC decay was also unchanged
suggesting that AppCH.sub.2ppA was not modulating the NMDA
component of EPSCs either (FIG. 5C).
[0152] In contrast, the literature shows that Ap.sub.4A induces the
inhibition of excitatory postsynaptic currents as well as
orthodromically evoked filed potentials..sup.14 These results have
also proven difficult to reproduce and are unreliable.
Experiment 2
[0153] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 8 mins,
pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (20
.mu.M) was applied. Then after 12 mins, Ap.sub.2CH.sub.2p.sub.2A
was applied.
[0154] It was found that using
pyridoxal-phosphate6-azophenyl-2',4'-disulphonic acid (PPADS)
completely abolished the blocking effect of AppCH.sub.2ppA on the
orthodromic spikes (FIG. 6). FIG. 6 shows, on the left, the time
course of the changes of amplitude in orthodromically induced
population, and on the right it shows the original traces of
population spikes (five-fold averaged) corresponding with points 1
(control) and 2 (Ap.sub.2CH.sub.2p2A/PPADS effect) in the time
course. Since PPADS is a well-known broadband P2-receptor family
antagonists.sup.15,16, this result suggested that the observed
AppCH.sub.2ppA effects could be mediated by a novel P2-family
receptor with unconventional pharmacology.
Experiment 3
[0155] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 9 mins .alpha.,.beta.-methylene-ATP
(1001M) was applied (FIG. 8).
Experiment 4
[0156] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. Over time, increasing amounts of ATPYS was
applied to the rat hippocampal slice (FIG. 9). Thus, 10 .mu.m of
ATPYS was applied after 10 mins; this was increased to 20 .mu.m
after 15 mins; to 50 .mu.m after 21 mins; and to 100 .mu.m after 29
mins.
[0157] Experiments 3 and 4 are agonist experiments using known
agonists of P2X and/or P2Y-family receptors. However,
.alpha.,.beta.-methylene-ATP (FIG. 8) and ATP.gamma.S (FIG. 9) were
unable to inhibit orthodromically evoked field potentials in the
same way as AppCH.sub.2ppA.
[0158] The only effect observed was a weak, slowly developing
inhibition at very high nucleotide concentrations (100 .mu.M).
Given the high concentrations of agonists used, such effects are,
at best, non-specific. Therefore, it appears that AppCH.sub.2ppA
effects are unlikely to be mediated by the main P2X or P2Y-family
receptors. This is perhaps surprising in view of the known capacity
of Ap.sub.nAs to act as agonists of P2X.sub.1-4, P2Y.sub.1,
P2Y.sub.2 and P2Y.sub.4 receptors in neurological
tissue.sup.15.
Experiment 5
[0159] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured (FIG. 10. After 10 mins, diinosine
tetraphosphate (Ip.sub.4I) (20 .mu.M) was applied. Then after 20
mins, Ap.sub.2CH.sub.2p.sub.2A (7.4 .mu.M) was applied.
Experiment 6
[0160] The time course of the changes of amplitude in
antidromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured (FIG. 11). After 10 mins, diinosine
tetraphosphate (Ip.sub.4I) (20 .mu.M) was applied. Then after 16
mins, Ap.sub.2CH.sub.2p.sub.2A (7.4 .mu.M) was applied.
[0161] The possibility that the effects of AppCH.sub.2ppA could be
mediated by the P4-dinucleotide receptor previously
identified.sup.17 on rat brain synaptic terminals was evaluated in
Experiments 5 and 6. However, it was found that addition of even
high concentrations of the P4 antagonist diinosine tetraphosphate
(Ip.sub.4I) (20 .mu.M) did hot alter the effect of AppCH.sub.2ppA
on the changes of amplitude in orthodromically induced population
spikes (FIG. 10). In addition, it was found that addition of
Ip.sub.4I (20 .mu.M) did not cause AppCH.sub.2ppA to have an effect
on the changes of amplitude in antidromically induced population
spikes (FIG. 11). These results completely rule out the possibility
of P4-dinucleotide receptor mediation. Thus, these results suggest
that a new P2-family receptor could be mediating the observed
AppCH.sub.2ppA effects.
Experiment 7
[0162] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 10 mins, bicuculine (50 .mu.M) was
applied. Then after 20 mins, Ap.sub.2CH.sub.2p.sub.2A (7.4 .mu.M)
was applied.
[0163] The AppCH.sub.2ppA induced inhibition of orthodromically
evoked field potentials was unaffected by the presence of
bicuculine.
Experiment 8
[0164] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 10 mins, hexamethonium (100 .mu.M)
was applied. Then after 20mins, Ap.sub.2CH.sub.2p.sub.2A (7.4
.mu.M) was applied.
[0165] The AppCH.sub.2ppA induced inhibition of orthodromically
evoked field potentials was unaffected by the presence of
hexamethonium.
Experiment 9
[0166] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured (FIG. 7). After 10 mins, strychnine (500 nM)
was applied. Then after 20 mins, Ap.sub.2CH.sub.2p.sub.2A (7.4
.mu.M) was applied.
[0167] The AppCH.sub.2ppA induced inhibition of orthodromically
evoked field potentials was unaffected by the presence of
strychnine.
Experiment 10
[0168] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 10 mins, cyclopentyl teophylline
(CPT) (1 .mu.M )was applied. Then after 26 mins,
Ap.sub.2CH.sub.2p.sub.2A (7.4 .mu.M) was applied.
[0169] The AppCH.sub.2ppA induced inhibition of orthodromically
evoked field potentials was eliminated by the presence of CPT.
[0170] Experiments 7, 8, 9 and 10 investigate the location of the
receptor that mediates the AppCH.sub.2ppA effects. It can be
deduced that the precise inhibition of orthodromic spikes must
arise from the modulation of postsynaptic neuron excitability alone
because AppCH.sub.2ppA does not modulating synaptic transmissions
at all. Furthermore as antidromically evoked field potentials are
not modulated by AppCH.sub.2ppA then the site of modulation must be
located in postsynaptic CA1 dendrites. The dendrites of CA1
pyramidal neurons are well known as an important target for
cortical modulation mediated via numerous receptors including
cholinergic and GABAergic receptors. Accordingly, these experiments
used known antagonists of .gamma.-aminobutyric acid (GABA)
(Experiment 7), muscarinic (Experiment 8) and glycine receptors
(Experiment 9). However, none of these antagonists affected the
AppCH.sub.2ppA induced inhibition of orthodromically evoked field
potentials. By contrast, the administration of CPT, an A1 adenosine
receptor antagonist, was seen to eliminate the effect of
AppCH.sub.2ppA (FIG. 7), thereby suggesting that AppCH.sub.2ppA
effects are mediated instead by A1 adenosine receptor activation
downstream of PPADS-sensitive P2 receptor activation.
[0171] This suggestion is supported by the fact that the
administration of adenosine (5 .mu.M) to hippocampal slices has
been shown previously to inhibit orthodromic spikes.sup.18 in a
similar fashion to AppCH.sub.2ppA. However, adenosine
administration was also shown to diminish EPSC amplitudes,.sup.19
in direct contrast with the effect observed following
AppCH.sub.2ppA administration (FIG. 5C). Therefore, AppCH.sub.2ppA
mediated effects are altogether more selective than adenosine
alone.
Experiment 11
[0172] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 5 mins,
2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, PTIO, (1 mM)
was applied (FIG. 12). Then after 11 mins, Ap.sub.2CH.sub.2P.sub.2A
(2.5 .mu.M) was applied.
[0173] The use of PTIO reduced the extent of
AppCH.sub.2ppA-mediated inhibition of orthodromically evoked field
potentials by over 50% (FIG. 12).
[0174] Nitric oxide (NO) has been shown to mediate adenosine
outflow in response to P2-receptor activation..sup.20 Thus, the use
of PTIO, a known NO specific scavenger, would be expected to affect
the AppCH.sub.2ppA-mediated inhibition of orthodromically evoked
field potentials if this involves a P2 receptor. The observed
reduction is consistent with a P2 receptor having a direct role in
this case. Thus, it may be postulated that AppCH.sub.2ppA-mediated
effects proceed by a pathway that links PPADS-sensitive P2 receptor
activation, resulting from the binding of AppCH.sub.2ppA, with the
production of NO that subsequently stimulates the intracellular
synthesis of adenosine leading to exclusive postsynaptic A1
receptor activation.
Experiment 12
[0175] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured. After 10 mins, adenosine deaminase (approx.
2 U/ml) was applied. Then after 20 mins, Ap.sub.2CH.sub.2p.sub.2A
(7.4 .mu.M) was applied.
[0176] The AppCH.sub.2ppA induced inhibition of orthodromically
evoked field potentials was eliminated by the presence of adenosine
deaminase.
[0177] Nucleoside-activated receptors have been observed to bring
about presynaptic inhibition of glutamate release in hippocampal
neurons in an earlier study..sup.21 This process is mediated by
so-called P3 receptors (P2Y-theophylline-sensitive receptors) and
has some similarities to the observed AppCH.sub.2ppA effects.
However, the fact that AppCH.sub.2ppA effects were eliminated by
adenosine deaminase is inconsistent with a P3 mechanism, indicating
that AppCH.sub.2ppA effects are not mediated through a similar
pathway.
Experiment 13
[0178] The time course of the changes of amplitude in
orthodromically induced population spikes in CA1 zone of rat
hippocampal slices prepared in accordance with the general
procedure was measured before and after the addition of
Ap.sub.2CH.sub.2p.sub.2A (2.5 .mu.M) at 22.degree. C. (FIG. 13) and
at 36.degree. C. (FIG. 14).
[0179] The temperature dependence of this effect is consistent with
a signalling pathway that involves the diffusion of small molecule
mediators such as adenosine.
Experiment 14
[0180] Modulation of NMDA-receptor mediated current by
non-hydrolysible analogues of diadenosine polyphosphate.
[0181] Diadenosine polyphosphates are natural compounds that can
play a neurotransmitter role in the synaptic terminals of the
central nervous system. Here we demonstrate that non-hydrolysible
analogue of diadenosine polyphosphates AppCH.sub.2ppA may affect
the functioning of NMDA-receptor-mediated channels. In isolated
hippocampal pyramidal neurons, AppCH2ppA applied at low micromolar
concentrations increased the amplitude of the NMDA-activated
current in a concentration-dependent manner. These effects of
AppCH.sub.2ppA were eliminated in the presence of purine P2
receptors antagonists PPADS and reactive blue, suggesting that
effects of AppCH.sub.2ppA are mediated by activation of purine
receptors. The effects of AppCH.sub.2ppA were removed in the
presence EDTA, indicating that some of bivalent cations, tonically
present in extracellular solution in the trace amount are involved
in the observed effects downstreams of the activation of
P2-receptors. Furthermore the effects of AppCH.sub.2ppA were
abolished after pre-treatment of neurons with the non-specific
inhibitor of tyrosine protein kinase inhibitor genestein. These
data taken together allow suggesting that AppCH.sub.2ppA
potentiated NMDA-currents is due to P2 receptor-dependent
activation of tyrosine kinase via reducing the tonic inhibition of
NMDA receptors by some of bivalent cations, most probably
Zn.sup.2+.
[0182] The results are shown in FIGS. 15 and 16.
[0183] FIG. 15 shows a modulation of NMDA receptor-activated
currents recorded in isolated hippocampal pyramidal neurons by
AppCH2ppA (1 .mu.M) is mediated by purine P2 receptors.
[0184] NMDA-receptor-activated currents were evoked by 1-2 sec long
co-application of aspartate (ASP) (1 mM) and glycine (10 .mu.M).
Vh=-100 mV in Mg.sup.2+ free solution.
[0185] (a) Representative traces of NMDA-activated current in
control, in the presence and after wash-out of AppCH.sub.2ppA.
[0186] (b) Statistics for the effects of AppCH.sub.2ppA and other
purine agonists; ADP(1 .mu.M), UTP(1 .mu.M), UDP (1 .mu.M) on the
peak amplitude of NMDA-current,
[0187] (c) Inhibition of AppCH.sub.2ppA--mediated enhancement of
NMDA-currents by non-specific purine P2 receptor antagonist
PPADS.
[0188] (d) statistics for the effects of AppCH.sub.2ppA on the peak
amplitude of NMDA-current in control conditions and in the presence
of P2 antagonists PPADS.
[0189] (e) Inhibition of AppCH.sub.2ppA--mediated effects by P2Y
receptor antagonists Reactive blue (RB).
[0190] FIG. 16 shows a modulation of NMDA receptor-activated
currents by AppCH.sub.2ppA is mediated by relief from tonic
inhibition by bivalent cations.
[0191] (a) Potentiation of NMDA--activated currents is eliminated
in the presence of chelator of bivalent cations EDTA.
Representative traces of NMDA-activated current in control, in the
presence of EDTA and in the presence of AppCH.sub.2ppA and
EDTA.
[0192] (b) Statistics for the effects of AppCH.sub.2ppA on the peak
amplitude of NMDA-current in control conditions and after
pre-application of EDTA.
[0193] (c) Inhibition of AppCH.sub.2ppA--mediated enhancement of
NMDA-currents by non-specific inhibitor of tyrosine protein kinase
genestein.
[0194] (d) statistics for the effects of AppCH.sub.2ppA on the peak
amplitude of NMDA-current in control conditions and after
pre-treatment of genestein.
MATERIALS AND METHODS
Materials
[0195] All the chemicals for intra- and extra-cellular solutions
were purchased from Sigma Chemical Co. (St. Louis, Mo., USA).
Cell Preparation
[0196] Wistar rats (12-17-days old) were decapitated under ether
anaesthesia and the hippocampus (or cerebellum) was removed. It was
cut into slices (300-500 .mu.m) in a solution containing (in mM):
150 NaCl; 5 KCl; 1.25 NaH.sub.2PO.sub.4; 26 NaHCO.sub.3; 1.1
MgCl.sub.2; 10 glucose; pH 7.4. Then the slices were incubated for
10 min at 32.degree. C. with 0.5 mg/ml of protease (type XXIII)
from Aspergillus oryzae. Single pyramidal cells from CA1 and CA3
stratum pyramidale layers were isolated by vibrodissociation
locally in the stratum pyramidale CA3 and CA1 hippocampal pyramidal
neurons were identified by their characteristic form and partially
preserved dendritic arborisation.
[0197] After isolation the cells were usually suitable for
recordings for 2-4 h. Throughout the entire procedure the solutions
with the slices were continuously saturated with 95% O.sub.2 and 5%
CO.sub.2 gas mixture to maintain pH 7.4. The tested substances were
dissolved in DMSO to a stock concentration of 10 mM and kept frozen
at -40.degree. C. in daily aliquots. The substances were dissolved
in external saline to their final concentration immediately before
the experiments.
Current Recordings
[0198] NMDA-activated currents in isolated neurons were induced by
the step application of aspartate (1 mM) and glycine (1 mM) in the
"concentration clamp" mode (Krishtal et al., 1983), using the
computerized "Pharma-Robot" set-up (Pharma-Robot, Kiev). This
equipment allows a complete change of saline within 15 ms.
Transmembrane currents were recorded using a conventional
patch-clamp technique, in the whole-cell configuration. Patch-clamp
electrodes were pulled with a horizontal puller (Sutter
Instruments) and had an internal tip diameter between 1.4 and 1.8
.mu.m and a tip resistance between 2.5 and 5 MOm. The intracellular
solution contained (in mM): 70 Tris-PO.sub.4; 5 EGTA; 40 TEA-Cl
(tetraethylammonium chloride); 30 Tris-Cl; 5 Mg-ATP; 0.5 GTP; pH
7.2. The composition of extracellular solution was (in mM):. 130
NaCl; 3 CaCl.sub.2; 5 KCl; 2 MgCl.sub.2; 10 HEPES-NaOH; 0.1 .mu.M
TTX; pH 7.4. Recording of the currents was performed using
patch-clamp amplifiers (DAGAN, USA). Transmembrane currents were
filtered at 3 kHz, stored and analysed with an IBM-PC computer
using homemade software. NMDA responses were recorded with a 3 min
interval. All experiments were performed at room temperature
(19-24.degree. C.).
Experiment 15
Antinociceptive Activity of AppCH.sub.2ppA
[0199] Experimental Procedure:
[0200] CFA-Induced Thermal Hyperalgesia. Unilateral inflammation
was induced by injecting 100 .mu.l of 50% solution of CFA (Sigma)
in physiological saline into the plantar surface of the right hind
paw of the rat. The hyperalgesia to thermal stimulation was
determined 48 h after CFA injections using the same apparatus as
described below for the noxious acute thermal assay.
[0201] Thermal sensitivity: On each day following mechanical
testing, rats will be placed in a thermal testing apparatus
(Plantar test, Ugo Basile, Italy), in which they will be free to
move. After 30 min of acclimatization, a constant-power IR stimulus
will be focused through the glass base on to the sole of the foot,
and the latency for reflex foot withdrawal will be recorded
automatically via a photoelectric monitor as previously described
Hargreaves et al. 1988.
[0202] In each test session, each rat (from 6 tested rats) was
tested in three sequential trials at approximately 15 min
intervals.
Results
[0203] Antinociceptive activity of AppCH.sub.2ppA. To characterize
the nociceptive activity of AppCH.sub.2ppA the effects of this
compound was evaluated in CFA-induced thermal hyperalgesia animal
model after s.c. administration. AppCH.sub.2ppA was evidently
potent in reducing thermal hyperalgesia (FIG. 17).
[0204] After 48 h of inflammation induced by the intraplantar
administration of CFA, AppCH.sub.2ppA fully blocked thermal
hyperalgesia (FIG. 17). The antinociceptive effects of
AppCH.sub.2ppA were specific to the injured paw, as the paw
withdrawal latencies for the uninjured paw were less effectively
altered by AppCH.sub.2ppA at the doses tested. The antinociceptive
effects of AppCH.sub.2ppA in the injured paw were delayed in onset
and appeared after 3 hours after injection.
[0205] FIG. 17--AppCH.sub.2ppA increases paw withdrawal latencies
48 h after intraplantar administration of CFA. Responses (paw
withdrawal latencies (mean.+-.SEM)) in control and CFA-injected
paw.
[0206] AppCH.sub.2ppA (50 .mu.mol/kg s.c.), attenuates CFA-induced
thermal hyperalgesia in the rat
[0207] FIG. 18--AppCH.sub.2ppA increases paw withdrawal latencies
48 h in contra lateral (non-inflamed) paw. Responses (paw
withdrawal latencies (mean.+-.SEM)) in control and contra lateral
paw.
[0208] AppCH.sub.2ppA (50 .mu.mol/kg s.c.), attenuates thermal
hyperalgesia in the rat.
[0209] * P<0.05 CFA vs CFA+AP4 50 uM
[0210] $ P<0.05 CFA vs control
[0211] ** P<0.05 CFA vs contra latheral paw CFA+AppCH2ppA 50
uM
[0212] P control vs CFA 1.58E-12
[0213] P CFA vs CFA+AP4 5 uM 0.000314
[0214] P CFA vs CFA+AP4 50 uM 0.000501
[0215] P CFA vs contra latheral paw CFA+AP4 50 uM 0.0043
[0216] P CFA vs contra latheral paw CFA+AP4 50 uM 0.000335
[0217] P CFA vs contra latheral paw CFA+AP4 50 uM 3.67E-06
[0218] P CFA vs contra latheral paw CFA+AP4 50 uM 1.53E-07
[0219] P CFA vs contra latheral paw CFA+AP4 50 uM 6.54E-05
Apparatus Description
[0220] 7370-Plantar Test
Measurement of Hyperalgesia to Thermal Stimulation in Unrestrained
Animals
[0221] Featuring:
[0222] Automatic detection of the behavioral end point
[0223] Validity unaffected by repeated testing
[0224] Greater bioassay sensitivity than other thermal or
mechanical tests
[0225] Each animal can serve as its own control
[0226] The Instrument basically consists of:
[0227] a Movable I.R. (infra-red) Source
[0228] a Glass Pane onto which the Rat Enclosure is located
[0229] a Controller
[0230] A 3-compartment enclosure has been provided to speed up the
test when a number of animals is involved. In each compartment the
animal is unrestrained.
[0231] After the acclimation period, the I.R. Source placed under
the glass floor (see the picture) is positioned by the operator
directly beneath the hind paw. A trial is commenced by depressing a
key which turns on the I.R. Source and starts a digital solid state
timer.
[0232] When the rat feels pain and withdraws its paw, the sudden
drop of reflected radiation switches off the I.R. Source and stops
the reaction time counter.
[0233] The withdrawal latency to the nearest 0.1 s is
determined.
Calibration Radiometer
[0234] Each Plantar Test is accurately calibrated via an I.R.
Radiometer to make sure that its I.R. source delivers the same
power flux (expressed in mW per square cm) and hence a nociceptive
stimulus of the same intensity.
[0235] The end user should consider this extremely useful
accessory, the Heat-Flow I.R. Radiometer Cat 37300, a battery
operated, self sufficient instrument complete with I.R. probe,
digital meter and adaptors for the Tail Flick and Plantar Test, all
parts neatly lodged in a sturdy plastic case with punched foam
lining.
[0236] The 37300 Radiometer enables the experimenter to:
[0237] i) Check (and adjust if necessary) the I.R. emission. In
fact, the I.R. output of the Plantar Test may in the course of
one-two years undergo to 2-3% reduction, due to dust gathered on
the optics, blackening of the I.R. bulb, accidental knocks, ageing
of components due to thermal cycles, etc.
[0238] Moreover, in case the bulb is replaced or the electronics
serviced, output alteration of more significant magnitude, say,
8-10%, may take place.
[0239] ii) Make sure that two or more Plantar-Test units deliver
thermal nociceptive stimuli of exactly the same intensity. Balance
them, if necessary.
[0240] iii) Know the I.R. energy (1 mW for the duration of is
corresponds to 1 mJ) in absolute terms, a useful datum to compare
with any equal or different method/instrument described in the
literature.
BIBLIOGRAPHY
[0241] Method Paper:
[0242] K. M. Hargreaves, R. Dubner, F. Brown, C. Flores and J.
Joris: "A New and Sensitive 5 Method for Measuring Thermal
Nociception in Cutaneous Hyperalgesia." Pain 32: 77-88, 1988.
[0243] Additional Papers:
[0244] K. M. Hargreaves, R. Dubner and J. Joris: "Peripheral Action
of Opiates in the Blockade of Carrageenan-Induced Inflammation"
Pain Research and Clinical Management. Vol. 3. Elsevier Science
Publishers, Amsterdam: 55-60, 1988
[0245] G. Benneth and Y. K. Xie: "A Peripheral Neuropathy in Rat
that Produces Disorders of Pain Sensation Like Those Seen in Man"
Pain 33: 87-107, 1988.
[0246] M. Iadarola and G. Draisci: "Elevation of Spinal Cord
Dynorphin mRNA Compared to Dorsal Root Ganglion Peptide mRNAs
During Peripheral Inflammation" In: The Arthritic Rat as a Model of
Clinical Pain? by J. Besson and G. Guilbaud (eds.) Elsevier Press,
Amsterdam: 173-183, 1988.
[0247] A. Costello and K. M. Hargreaves: "Suppression of
Carrageenan-Induced Hyperalgesia. Edema and Hyperthermia by a
Bradykinin Antagonist" European J. Pharmacol., 1989.
[0248] K. M. Hargreaves, R. Dubner and A. Costello: "Corticotropin
Releasing Factor (CRF) has a Peripheral Site of Action for
Antinociception" European J. Pharmacol., 1989.
[0249] J. Hylden, R. Nahin, R. Traub and R. Dubner: "Expansion of
Receptive Fields of Spinal Lamina I Protection Neurons in Rats with
Unilateral Adjuvant-induced Inflamma-tion: The Contribution of
Central Dorsal Horn Mechanisms" Pain 37: 229-244, 1989.
[0250] In addition, more than 30 abstracts using this device have
been presented at U.S. (eg. Society for Neuroscience) and
International (e.g., International Association for the Study of
Pain) scientific meeting
[0251]
http://www.ugobasile.com/site/manuals/condensed_catalogue.pdf
[0252] http://www.ugobasile.com/site/product2.asp?ID=3
[0253] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in chemistry, biology or related
fields are intended to be within the scope of the following
claims
REFERENCES
[0254] 1. Pintor, J.; Gualix, J.; Miras-Portugal, M. T. Mol.
Pharm., 1997, 51, 277-284.
[0255] 2. Oaknin, S.; Rodriguez-Ferrer, C. R.; Aguilar, J. S.;
Ramos, A.; Rotlian, P. Neurosci. Lett., 2001, 309, 177-180.
[0256] 3. Ap.sub.4A and other dinucleoside polyphosphates, Ed. A.
G. McLennan, CRC Press, Boca Raton, Fla., 1992.
[0257] 4. Plateau, P.; Blanquet S., Adv. Micro. Physiol., 1994, 36,
81.
[0258] 5. Wang, Y., Chang, C. F., Morales, M., Chiang, Y. H.,
Harvey, B. K., Su, T. P., Tsao, L. I., Chen, S., Thiemermann, C. J.
Neuroscience 2003, 23, 7958-65.
[0259] 6. Ortiz, B.; Sillero, A.; Gunther Sillero, M. A.; Eur. J.
Biochem., 1993, 212, 263.
[0260] 7. Theoclitou, M.-E.; Wittung, E. P. L.; Hindley, A. D.;
El-Thaher, T. S. H.; Miller, A. D. J. Chem. Soc., Perkin Trans. 1,
1996, 2009-2019.
[0261] 8. McLennan, A. G. Pharmacol Ther, 2000, 87, 73-89.
[0262] 9. Wright, M., Tanner, J. A., and Miller, A. D. Anal
Biochem, 2003, 316, 135-138.
[0263] 10. Guranowski, A. Pharmacol Ther, 2000, 87, 117-39.
[0264] 11. Theoclitou, M. E.; El-Thaher, T. S. H.; Miller, A. D. J.
Chem. Soc., Chem. Commun., 1994, 659-661.
[0265] 12. Wright, M.; Miller, A. D. Bioorg. Med. Chem. Lett.,
2004, 14, 2813-2816.
[0266] 13. Ng, K. E.; Orgel, L. E. Nucleic. Acids. Res., 1987, 15
(8), 3573-3580.
[0267] 14. Klishin, A.; Lozovaya, N.; Pintor, J.; Miras-Portugal,
M. T.; Krishtal, O. Neuroscience, 1994, 58, 235-6.
[0268] 15. Pintor, J.; Diaz-Hernandez, M.; Gualix, J.;
Gomez-Villafuertes, R.; Hernando, F.; Miras-Portugal, M. T.
Pharmacol Ther, 2000, 87, 103-15.
[0269] 16. Bianchi, B. R.; Lynch, K. J.; Touma, E.; Niforatos, W.;
Burgard, E. C.; Alexander, K. M.; Park, H. S.; Yu, H.; Metzger, R.;
Kowaluk, E.; Jarvis, M. F.; van Biesen, T. Eur J Pharmacol, 1999,
376, 127-38.
[0270] 17. Pintor, J.; Miras-Portugal, M. T. Br J Pharmacol, 1995,
115, 895-902.
[0271] 18. Greene, R. W.; Haas, H. L. Prog Neurobiology, 1991, 36,
329-41.
[0272] 19. Klishin, A.; Lozovaya, N.; Krishtal, O. Neuroscience,
1995, 65, 947-53.
[0273] 20. Juranyi, Z.; Sperlagh, B.; Vizi, E. S. Brain Res, 1999,
823, 183-90.
[0274] 21. Mendoza-Fernandez, V.; Andrew, R. D.; Barajas-Lopez, C.
2000 J Pharmacol Exp Ther, 2000, 293, 172-9.
[0275] 22. Rudolphi K; Schubert P; Parkinson F. E.; Fredholm B. B.
Trends Pharmacol Sci. 1992, 13, 439-445.
[0276] 23. Knutsen L. J. S.; Murray T. F. Adenosine and ATP in
epilepsy. In Purinergic Approaches in Experimental Therapeutics.
Eds. Jacobson K. A,; Jarvis M. F. Wiley-Liss, New York, 1997, pp.
423-447.
[0277] 24. Dragunow M. Adenosine and Adenine Nucleotides as
Regulators of Cellular Function. Ed. Phillis J. W., Boca Raton,
Fla., CRC Press, 1991, 367-379.
[0278] 25. IJzerman A. P.; van der Wenden N. M. Purinergic
Approaches in Experimental Therapeutics Ed. Jacobson, K. A.;
Jarvis, M. F., Wiley-Liss, Inc. New York, 1997, 129-148.
[0279] 26. Firestein G. Drug Dev. Res. 1996; 39: 371-376.
[0280] 27. Ferre S.; Fredholm B. B.; Morelli M.; Popoli P.; Fuxe K.
Trends Neurosci 1997; 20:482-487.
[0281] 28. Carley D.; Radulovacki M.; Purinergic Approaches in
experimental Therapeutics. Eds. Jacobson K. A.; Jarvis M. F.,
Wiley-Liss, New York, pp.515-526.
[0282] 29. Sawynok J. Eur J Pharmacol 1998; 347:1-11.
[0283] 30. Cook S. P.; Vulchanova L.; Hargreaves K. M.; Elde R.;
McCleskey E. W. Nature 1997; 387:505-508.
[0284] 31. Burnstock G. A. Lancet 1996, 347, 1604-1605.
[0285] 32. Vulchanova L.; Arvidsson U.; Riedl M.; Wang J.; Buell
G.; Surprenant A.; North R. A.; Elde R.; Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 8063-8067.
[0286] 33. Keil G. J.; DeLander G. E. Life Sci 1992;
51:L171-L176.
[0287] 34 Kowaluk E. A.; Bhagwat S. S.; Jarvis M. F. Curr.
Pharmaceut. Design 1998; 4: 403-416.
[0288] 35. Poon A.; Sawynok J. Pain 1998; 74:235-245.
[0289] 36. Lee Y. W.; Yaksh T. L. J Pharmacol Exp Ther 1996; 277:
1642-1648.
[0290] 37. Segerdahl M.; Ekblom A.; Sandelin K.; Wickman M.;
Sollevi A. Anesth Analg 1995; 80:1145-1149.
[0291] 38. Sollevi A. Acta Anaesthesiol Scand Suppl 1997;
110:135-136.
[0292] 39. Woolf, C. J. Textbook of Pain, 3rd Ed. Eds Wall, P.;
Melzack, R. D., Churchill Livingstone, Edinburgh, 1994,
101-112.
[0293] 40. Fukunaga A. F. Purinergic Approaches in Experimental
Therapeutics. Eds Jacobson K. A., Jarvis M. F., Wiley-Liss, New
York, 1997, pp. 471-493.
* * * * *
References